Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

Interaction of mitochondrial creatine kinase with model membranes. A monolayer study

The interaction of mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi-CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi-CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligomeric form of Mi-CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi-CK was able to interact equally well with both inner and outer mitochondrial membranes.     

Heterogeneity of mitochondrial creatine kinase

The heterogeneity of cardiac sarcomeric mitochondrial creatine kinase (creatine N-phosphotransferase, EC 2.7.3.2, sMi-CK), namely, brain ubiquitous Mi-CK (uMi-CK) and an atypical Mi-CK detected in the serum of a patient with ovarian cancer, was studied by isoelectric focusing. These Mi-CKs were found to be slightly different from each other with respect to their pIs under the examined conditions. The atypical Mi-CK was found to be an atypically oxidized form of uMi-CK. Results suggest that these heterogeneities of Mi-CK are caused by the genotypes, structures, biological functions and metabolism/dissimilation of Mi-CKs in the mitochondria and intravascular circulation.     

Mitochondrial creatine kinase - a square protein

The recently determined structure of octameric mitochondrial creatine kinase has provided new insights into the functioning of this enzyme and its role in channelling energy from the mitochondria to the cytoplasm. Creatine kinase, a member of the family of guanidino kinases, is structurally similar to glutamine synthetase, suggesting a possible evolutionary link between both protein families     

Mitochondrial creatine kinase in human health and disease

Mitochondrial creatine kinase (MtCK), together with cytosolic creatine kinase isoenzymes and the highly diffusible CK reaction product, phosphocreatine, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. Mitochondrial proteolipid complexes containing MtCK form microcompartments that are involved in channeling energy in form of phosphocreatine rather than ATP into the cytosol. Under situations of compromised cellular energy state, which are often linked to ischemia, oxidative stress and calcium overload, two characteristics of mitochondrial creatine kinase are particularly relevant: its exquisite susceptibility to oxidative modifications and the compensatory up-regulation of its gene expression, in some cases leading to accumulation of crystalline MtCK inclusion bodies in mitochondria that are the clinical hallmarks for mitochondrial cytopathies. Both of these events may either impair or reinforce, respectively, the functions of mitochondrial MtCK complexes in cellular energy supply and protection of mitochondria form the so-called permeability transition leading to apoptosis or necrosis.     

Study on heart mitochondrial creatine kinase using a cross-linking bifunctional reagent. II. The binding sites for the creatine kinase octamer on mitochondrial membranes have different properties

Beef heart mitochondria suspended in 0.25 M sucrose were treated with 0.3% glutaraldehyde (GA). The membranes were disintegrated by ultrasonication in 0.25 M KCl and precipitated by centrifugation. The relative amount of the membrane-bound mitochondrial creatine kinase (CKm) does not depend on the time course of membrane disruption. The enzyme is not removed by repeated washing of the pellet. It is concluded that this part of CKm is cross-linked to mitochondrial membranes. The maximum amount of the enzyme capable of cross-linking to the membrane with an increase in GA concentration or incubation time makes up to about 50% of the total CKm activity present in the mitochondria. It is concluded also that the CKm binding sites differ with respect to their environment.     

Expression of the mitochondrial creatine kinase genes

Mitochondrial Creatine Kinase (MtCK) is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier, creatine, and exists in mammals as two isoenzymes encoded by separate genes. In rats and humans, sarcomere-specific MtCK (sMtCK) is expressed only in skeletal and heart muscle, and has 87% nucleotide identity across the 1257 bp coding region. The ubiquitous isoenzyme of MtCK (uMtCK) is expressed in many tissues with highest levels in brain, gut, and kidney, and has 92% nucleotide identity between the 1254 bp coding regions of rat and human. Both genes are highly regulated developmentally in a tissue-specific manner. There is virtually no expression of sMtCK mRNA prior to birth. Unlike cytosolic muscle CK (MCK) and brain CK (BCK), there is no developmental isoenzyme switch between the MtCKs. Cell culture models representing the tissue-specific expression of either sMtCK or uMtCK are available, but there are no adequate developmental models to examine their regulation. Several animal models are available to examine the coordinate regulation of the CK gene family and include 1) Cardiac Stress by coarctation (sMtCK, BCK, and MCK), 2) Uterus and placenta during pregnancy (uMtCK and BCK), and 3) Diabetes and mitochondrial myopathy (sMtCK, BCK, and MCK). We report the details of these findings, and discuss the coordinate regulation of the genes necessary for high-energy transduction.     

Cardiolipin mediates curcumin interactions with mitochondrial membranes

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.     

Mitochondrial creatine kinase and mitochondrial outer membrane porin show a direct interaction that is modulated by calcium

Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.     

Crystal structure of human ubiquitous mitochondrial creatine kinase

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.     

Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 +/- 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.     

Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and "banana-shaped" structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.     

Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction = [ADP]·[CP][ATP]·[Cr] became equal to the apparent equilibrium constant (K _eq ^app) of this reaction. Subsequent excess of over the K _eq ^app was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after exceeded the K _eq ^app, but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in . Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although > K _eq ^app. The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.     

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.     

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Background  

The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).     

The quaternary structure of bovine heart mitochondrial creatine kinase

Crosslinking of subunits of the high molecular weight oligomer of bovine heart mitochondrial creatine kinase (CKm) by dimethyl suberimidate and subsequent electrophoresis in the presence of sodium dodecyl sulfate gives eight protein bands. An increase in the time course of the enzyme crosslinking reaction results in the protein accumulation in the high molecular weight bands. Evidence has been obtained suggesting that crosslinking involves only the intraoligomeric contact areas. It is concluded that bovine heart CKm is an octamer. Crosslinking of intersubunit contacts in the octameric form of the enzyme by various diimidates has been carried out. The data obtained suggest that within the octamer the CKm subunits have a quasispherical rather than planar arrangement. This finding is supported by electron microscopy data.